In the present study we evaluated the effects of chronic exposure to sounds at 22 kHz during pregnancy on the central serotonergic and behavioral parameters in Wistar rat dams after the suckling period and on their male rat offspring. In addition, we also assessed the effects of an acute 22 kHz sound, associated with the chronic intrauterine exposure, on the emotional responses of adult offspring. The primary hypothesis was that experiencing 22 kHz stimuli during an early stage of development would interfere with brain serotonergic parameters and, later, with the adult rat’s defensive responses. The corollary question was whether a 22 kHz sound exposure would differentially affect inhibitory avoidance and escape responses and central serotonergic parameters. Female rats were divided into four groups: non-pregnant control; non-pregnant chronic exposure; pregnant control; and pregnant chronic exposure. Male offspring were divided into four groups: chronic intrauterine sound exposure; acute sound exposure in adulthood; chronic intrauterine exposure with acute exposure in adulthood; and no exposure. Chronic sound exposure affected inhibitory avoidance and serotonergic parameters in female rats. For offspring, there was an interaction between chronic and acute sound exposure effects on inhibitory avoidance response but not on escape response. There were significant effects of chronic intrauterine exposure on serotonin turnover in the hippocampus and PFC of females. For offspring, the turnover was increased by chronic exposure only in PFC, and in amygdala it was increased by acute exposure. These results illuminate the potential of an early acoustic sound exposure for causing central serotonergic and emotional behavioral changes that can persist into later periods of life.
Rats can vocalize in the sonic and ultrasonic wide range. Their ultrasonic vocalizations (USV) range from 20 to about 100 kHz [
The primary hypothesis was that experiencing 22 kHz stimuli during an early stage of development would interfere with brain serotonergic parameters and, later, with the adult rat’s defensive responses. The corollary question was whether a 22 kHz sound exposure would differentially affect inhibitory avoidance and escape responses and central serotonergic parameters. The rationale of this idea is that a biological phenomenon known as programming can be triggered when an animal, e.g., a human [
Certain environmental conditions experienced during an early period of animal development induce programming. Maternal stress [
Some authors [
For instance, there is evidence that distinct brain structures and serotonin (5-HT) nerve fibers coming from the dorsal raphe nucleus (DRN) are involved in inhibitory avoidance and escape [
Forty-eight 3-month-old female Wistar rats were acquired from the vivarium of the Institute of Biological Sciences, Universidade Federal de Minas Gerais. Sixteen of them were separated into two groups (n = 8, each): Non-Pregnant Control (NPC) and Non-Pregnant chronic Exposure to 22 kHz sound (NPE). According to data obtained in previous experiments performed in our laboratory [
The female pregnant rats were placed in an acoustical isolation box and exposed to a 22 kHz sound 1hour/day from the 1st to the 19th day of gestation (PE group). The rats from NPE group received the same treatment as rats from PE group, that is, they were also exposed to a 22 kHz sound during the same period. All rats from the control groups (NPC and PC) were also placed in the acoustical box, as the same period as the treated rats, ex- cept that they were not exposed to the chronic 22 kHz sound. Upon removal from the acoustical isolation box, the female rats from each of the four groups were placed in individual cages. All rats from the PC and PE groups gave birth on the 21st day.
Offspring were inspected and only male pups remained with their mothers in the proportion of 6 - 8 per dam. After the 21-day lactation period, forty-eight offspring male rats (three from each litter) were randomly divided into four groups (n = 12, each): Control pups (C), Intrauterine Chronic Exposure pups (IE), Adult Acute Exposure pups (AE), Chronic Intrauterine and Acute Adult Exposure pups (IAE). The remaining rat offspring were removed from the present experiments. The rats from the four subgroups (total n = 48) were maintained in the animal house vivarium receiving water and food ad libitum for two months. AE and IAE groups were placed inside the acoustical isolation box and exposed to a 22 kHz sound for 1 hour. Rats from C and IE groups were placed into the acoustical isolation box for the same period of time as rats from AE and IAE groups but were not exposed to the acute 22 kHz sound. After the acute 22 kHz treatment the rats were submitted to the ETM tasks as described below. The dams were also submitted to this behavioral test two days after the lactation period.
Artificial tones of 22 kHz frequency were produced using the Matlab® software [
Defensive responses, inhibitory avoidance and escape related with anxiety and fear, were evaluated using the ETM behavioral model [
Memory test: after 72 hours of the training sessions, the rats were submitted to one more trial of inhibitory avoidance and one more trial of escape. For both tests, the ratio between the rat’s performances on the last session of training and the test 72 hours later was used as an index of memory performance.
All behavioral experiments were performed in a dimly lit and quiet room between 1 pm and 5 pm. The apparatus was cleaned with 20% ethanol solution between sessions.
The rats were decapitated 1 day after the end of behavioral tests. The brain was quickly removed and kept on ice. The thalamus, PFC, amygdala, PAG and hippocampus from hemispheres were immediately dissected according to the stereotaxic atlas [
The Kolmogorov-Smirnov test was used to verify the Gaussian assumption for the variables under study at a 0.05 significance level. The distribution for all variables was normal.
For both female and offspring rats, the performance on inhibitory avoidance and escape training tasks data were analyzed using ANOVA with repeated measures in the last element (2 × 2 × 3 factorial method). For females, the factors were pregnancy, chronic exposure to 22 kHz sound and trials (time), while for rat offspring the factors were chronic intrauterine 22 kHz exposure, acute adult 22 kHz exposure and trials (time). For both female and offspring, memory performance and biochemical data were analyzed using two-way ANOVA. All values are expressed as mean ± standard error (S.E.M.). Significance level was established at 5% for all tests.
Acquisition:
Memory performance: There were no significant effects of chronic 22 kHz exposure or pregnancy on female memory performance, tested in the inhibitory avoidance task, 72 hours after the training session (data not shown). There also was no interaction between these factors.
Acquisition:
Memory performance (data not shown): There were no significant effects for chronic intrauterine or for acute adult 22 kHz exposure on offspring memory performance, tested in the inhibitory avoidance task, 72 hours after the training session. There also was no interaction between these factors.
Acquisition:
There were no significant effects of any of the treatments, chronic 22 kHz exposure, pregnancy or trials. There also were no significant interactions between the factors.
Memory performance (data not shown): There were no significant effects of factors (chronic 22 kHz exposure, pregnancy and trials) or interactions between) them.
Acquisition:
Memory performance (data not shown): There were no significant effects of treatments either chronic intrauterine treatments or adult 22 kHz sound exposures on offspring memory performance tested in the escape task, 72 hours after the training session. There also was no interaction between these factors.
Although there were no significant effects of chronic 22 kHz exposure and pregnancy on 5-HT levels in any of the five brain areas evaluated, there was a significant interaction between them in the amygdala (F(3,10) = 5.81, P = 0.04). There were no significant interactions between factors on 5-HT levels in the other four regions evaluated (hippocampus, thalamus, PFC and PAG).
Neither chronic 22 kHz exposure nor pregnancy showed significant effects on 5-HIAA concentrations in any of the female brain areas analyzed. There also was no interaction between these factors.
Pregnancy had no effect on the 5-HT turnover ([5-HIAA]/[5-HT]) in any of the studied brain areas. There also was no effect of chronic 22 kHz exposure on 5-HT turnover in thalamus, amygdala and PAG. However, there were significant effects on 5-HT turnover in two areas, hippocampus [F(3,11) = 8.55, P = 0.01] and PFC [F(3,10) = 6.32, P = 0.03]. In addition, there were no interactions between the treatments on 5-HT turnover in any of the brain areas.
There were no significant effects of intrauterine exposure or acute adult exposure on 5-HT concentrations in any of the brain regions (hippocampus, thalamus, amygdala, PFC and PAD). In addition, there was no interaction between the two treatments.
Chronic intrauterine decrease and acute adult exposures increased the 5-HIAA concentrations in hippocampus. In this brain area there were significant effects of intrauterine 22 kHz exposure [F(3,40) = 7.17, P = 0.01] and acute adult exposure [F(3,40) = 5.25, P = 0.03] on 5-HIAA levels.
There also were significant interactions between treatments (chronic and acute exposure) on 5-HIAA concentration in hippocampus [F(3,40) = 8.05, P = 0.01] and amygdala [F(3,43) = 10.75, P < 0.01]. There were no effects of factors or interactions between them in the other evaluated regions.
Chronic exposure to 22 kHz sound increase 5-HT turnover in offspring PFC [F(3,42) = 11.28, P < 0.01], but there were no effects on any of the other brain areas. Acute adult exposure increase 5-HT turnover in amygdala [F(3,43) = 6.41, P = 0.01]. However, there was no effect of this treatment on 5-HT turnover in any the other stu- died brain regions. There also was no interaction between the factors for 5-HT turnover in any of the assessed brain areas.
The data of the present study show for the first time that the inhibitory avoidance response of female Wistar rats is affected by chronic exposure to a 22 kHz sound. Such results were not found in the escape response. In addition, this study demonstrates that chronic intrauterine exposure can also interfere with the inhibitory avoidance
[5-HT] | |||||||||
---|---|---|---|---|---|---|---|---|---|
Female | Offspring | ||||||||
NPC | NPE | PC | PE | C | IE | AE | IAE | ||
Hippocampus | 171.23 ± 18.82 | 166.96 ± 9.51 | 98.25 ± 14.18 | 202.75 ± 30.60 | 81.85 ± 23.65 | 81.85 ± 23.65 | 59.76 ± 26.94 | 57.05 ± 16.13 | |
Amygdala | 180.59 ± 13.41 | 151.35 ± 19.67 | 73.28 ± 9.84 | 151.57 ± 28.60 | 293.14 ± 75.56 | 182.35 ± 29.09 | 166.14 ± 54.12 | 154.07 ± 17.17 | |
PFC | 180.59 ± 13.41 | 311.31 ± 64.60 | 101.19 ± 30.42 | 275.65 ± 59.55 | 124.08 ± 20.40 | 90.40 ± 15.57 | 103.29 ± 22.86 | 82.66 ± 7.12 | |
PAG | 944.77 ± 37.60 | 159.69 ± 83.12 | 319.88 ± 55.13 | 353.58 ± 73.42 | 617.09 ± 14.46 | 434.57 ± 54.33 | 463.38 ± 59.63 | 544.91 ± 62.37 | |
Thalamus | 729.68 ± 32.15 | 224.54 ± 0.28 | 887.63 ± 74.82 | 1443.02 ± 42.16 | 186.25 ± 26.40 | 237.96 ± 33.19 | 238.21 ± 35.06 | 284.09 ± 39.82 | |
[5-HIAA] | |||||||||
Female | Offspring | ||||||||
NPC | NPE | PC | PE | C | IE | AE | IAE | ||
Hippocampus | 248.26 ± 30.61 | 182.26 ± 11.64 | 214.39 ± 7.05 | 207.68 ± 10.59 | 301.79 ± 27.23 | 293.44 ± 30.99 | 273.28 ± 28.92 | 532.13 ± 10.25 | |
Amygdala | 228.31 ± 51.53 | 128.83 ± 18.01 | 152.05 ± 14.79 | 179.95 ± 40.37 | 303.50 ± 41.38 | 194.53 ± 43.38 | 203.95 ± 15.46 | 423.68 ± 87.92 | |
PFC | 657.39 ± 62.29 | 598.35 ± 36.96 | 496.4 ± 65.18 | 976.31 ± 31.93 | 519.99 ± 43.09 | 561.14 ± 54.78 | 471.71 ± 42.57 | 618.97 ± 46.64 | |
PAG | 502.80 ± 15.85 | 658.38 ± 58.49 | 980.51 ± 23.96 | 842.13 ± 273.94 | 1403.81 ± 31.95 | 1785.30 ± 43.67 | 1311.33 ± 27.98 | 1392.11 ± 34.93 | |
Thalamus | 975.57 ± 15.41 | 1264.79 ± 22.27 | 745.03 ± 10.85 | 762.88 ± 36.53 | 706.06 ± 56.32 | 744.10 ± 54.43 | 746.06 ± 29.19 | 789.42 ± 31.54 | |
[5-HIAA]/[5-HT] | |||||||||
Female | Offspring | ||||||||
NPC | NPE | PC | PE | C | IE | AE | IAE | ||
Hippocampus | 1.54 ± 0.25 | 1.09 ± 0.07 | 2.24 ± 0.40 | 1.14 ± 0.21 | 7.36 ± 1.78 | 9.00 ± 1.93 | 10.13 ± 2.48 | 11.12 ± 1.37 | |
Amygdala | 1.34 ± 0.36 | 5.24 ± 0.20 | 2.14 ± 0.49 | 1.34 ± 0.18 | 1.49 ± 0.24 | 1.25 ± 0.24 | 1.89 ± 0.38 | 3.13 ± 1.33 | |
PFC | 4.13 ± 0.66 | 2.07 ± 0.40 | 5.18 ± 0.91 | 3.46 ± 0.64 | 4.98 ± 0.51 | 7.24 ± 0.80 | 5.64 ± 0.90 | 7.70 ± 0.46 | |
PAG | 0.48 ± 0.14 | 3.01 ± 1.42 | 2.39 ± 0.22 | 2.17 ± 0.39 | 2.80 ± 0.63 | 3.27 ± 0.72 | 3.73 ± 1.31 | 2.55 ± 0.53 | |
Thalamus | 4.26 ± 1.32 | 2.07 ± 0.40 | 2.54 ± 0.02 | 1.17 ± 0.67 | 4.60 ± 0.64 | 3.67 ± 0.43 | 3.67 ± 0.51 | 3.28 ± 0.56 |
Mean ± S.E.M. (ng/g of tissue) of serotonergic parameter levels in the brain areas; PFC: Prefrontal Cortex; PAG: Periaqueductal Gray; NPC: Non-Pregnant Control; NPE: Non-Pregnant Sound Exposure; PC: Pregnant Control; PE: Pregnant Sound Exposure; C: Control; IE: Intrauterine Sound Exposure; AE: Adult Sound Exposure; IAE: Intrauterine Adult Exposure; Females: There was a significant interaction between pregnancy and chronic 22 kHz sound exposure on 5-HT concentration in amygdale [F(3.10) = 5.81, P = 0.05]. There were also significant effects on 5-HT turnover in hippocampus [F(3.11) = 8.55, P = 0.01] and PFC [F(3.10) = 6.32, P = 0.03]. Offspring: There were significant effects of intrauterine 22 kHz exposure [F(3.40) = 7.17, P < 0.01] and acute adult exposure [F(3.40) = 5.25, P = 0.03] on 5-HIAA levels. There were also significant interactions between treatments on 5-HIAA concentrations in hippocampus [F(3.40) = 8.05, P < 0.01] and amygdale [F(3.43) = 10.75, P < 0.01]. There was a significant effect of chronic intrauterine exposure on 5-HT turnover in PFC [F(3.42) = 11.28, P < 0.01]. The other factors did not have significant effects.
(but not with escape) response of offspring rats confronted with an acute 22 kHz sound when they are adults. So, the previous chronic intrauterine experience interferes with the behavioral response to an acute 22 kHz exposure later in life. There is evidence that sounds in the range of 22 kHz frequency induce rat defensive behavior [
The pregnant and non-pregnant female rats that were previously exposed to the chronic 22 kHz sound exhi- bited decreased anxious behavior when tested in the ETM apparatus (decreased inhibitory avoidance latency). On the other hand, offspring submitted to a chronic 22 kHz sound during the intrauterine period did not show any defensive response change when assessed in the ETM. However, previous chronic intrauterine 22 kHz exposures followed by an acute 22 kHz exposure during adulthood also decrease inhibitory avoidance latency in ETM task. Similarly to dam performance, offspring escape response was not changed by 22 kHz sound applied in any of the experimental conditions used in the present study. These results agree with those obtained by other authors who, using pharmacological approaches, showed that 22 kHz USV in rats is an index of anxiety but not fear [
As the sound exposure used here was chronic, another hypothesis to be considered to explain the 22 kHz anxiolytic effects is that the dams and offspring could have learned, as a result of the repetitive exposures that the 22 kHz was not a sign of threat. This would explain the observation that over time their ETM inhibitory avoidance responses were lower compared with control rats. This hypothesis is supported by other authors who showed that repetitive sound exposure can induce habituation [
Three alternative hypotheses could be proposed for explaining the neurobiological and behavioral changes caused by two sequential 22 kHz stimuli applied in different periods of offspring development: 1) a direct effect of the first exposure on the offspring neuronal net, depending on the fact that the fetus would be able to “hear” or “perceive” the environmental sound. Compared to the effects on individuals not previously exposed, a second stimulus could produce a distinct response; 2) an indirect effect of the first exposure on the offspring neuronal net, as a result of a primary effect on the dams; or 3) both effects, direct and indirect, occurring simultaneously. Thus, a distinct response to a second 22 kHz sound exposure could take place because it changes specific components of the neuronal net, which would make part of the neurobiological mechanism responsible for the behavioral response to a specific kind of environmental stimulus. In other words, a specific change caused by preceding experiences―chronic intrauterine 22 kHz sound exposure―would affect the inhibitory avoidance to a subsequent similar stimulus―acute 22 kHz―faced by the individuals in later periods of life.
With regard to the first hypothesis about a direct effect of sound on offspring, some authors stated that fetuses of sheep are able to hear environmental sounds [
The observed effects of 22 kHz sound on ETM inhibitory avoidance and the absence of effect on escape behavior suggest that the neurobiological mechanisms related to anxiety and fear states, respectively, might be distinct. Some author shows that panic attack does not activate HPA axis [
In spite of the fact that there is evidence that serotonin uptake inhibitors decrease the number of 22 kHz ultrasonic rat vocalizations [
In the present study, the following serotonergic parameter results were obtained after a chronic exposure to a 22 kHz sound during pregnancy: 1) the female and the adult offspring 5-HT concentrations were not affected in any of the assessed brain areas (PFC, PAG, amygdala, thalamus and hippocampus), 2) the adult offspring 5- HIAA concentrations were decreased in hippocampus by chronic 22 kHz sound exposure and increased when both chronic and acute stimuli were associated, 3) the serotonergic turnover rate was decreased in the female PFC and hippocampus, and increased in the offspring PFC. Regarding acute exposure (adult offspring), similar effects were observed as follows: 1) there was no effect on 5-HT levels in any of the assessed brain areas, 2) 5-HIAA concentrations were decreased in the hippocampus, 3) the serotonergic turnover rate was increased in the amygdala, an effect that was not detectable when the previous chronic and acute stimuli were associated. In short, in terms of serotonergic activity assessed by the turnover rate, these findings suggest that chronic exposure to 22 kHz sound causes a serotonergic activity decrease in the hippocampus and in the PFC of females. On the other hand, acute exposure during adulthood increases serotonergic turnover rate in the amygdala. These data indicate the involvement of limbic brain system regions in the neurobiological processes (emotional learning) affected by 22 kHz sound.
The absence of significant effects of 22 kHz sound applied during pregnancy on 5-HT levels observed in the present study accords with reports from other authors, who also did not find effects of white noise exposure on offspring hippocampal 5-HT release [
In the present work the chronic intrauterine 22 kHz sound exposure affects female and offspring serotonergic turnover, increasing activity in the hippocampus. For female, this effect was also observed in PFC. For offspring, acute exposure increased serotonergic turnover in amygdala. The significant effects of chronic and acute treatments on 5-HIAA were observed in the hippocampus and an interaction of these treatments was found in the hippocampus and amygdala. It is important to emphasize that there were no significant effects on serotonergic parameters in the PAG and thalamus in any of the experimental conditions used in the present study. The observation that chronic and acute 22 kHz sound exposure affect inhibitory avoidance but not escape response agrees with the findings that these acoustic sounds affect serotonergic parameters of specific brain areas, such as the hippocampus and amygdala, but not the PAG. It is known that the defensive responses, inhibitory avoidance and escape, involve distinct serotonergic pathways that originate from the raphe nucleus and continue to the fore- brain and midbrain areas. The inhibitory avoidance is structured in the forebrain (e.g., amygdala, PFC), while the latter is largely integrated in the midbrain (mainly PAG) (review in [
The present results are in agreement with the assumption that anxiety and panic are qualitatively different emotions with distinct modulator mechanisms that are related to two defensive strategies, respectively: reaction to potential threat and to proximal menace. Besides illuminating the potential of an early acoustic sound exposure for causing central serotonergic and emotional behavioral changes that can persist into later periods of life, the present data also show that the 22 kHz sound exposure represents a useful tool for understanding the mechanism of a specific defensive behavior. That is, as the 22 kHz sound exposure affects inhibitory avoidance but not escape behavior, it might be relevant for clarifying physiological and molecular aspects of emotion.
The authors declare no conflicts of interest.
This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and Fa- pemig (Fundação de Amparo à Pesquisa do Estado de Minas Gerais). The authors thank Aparecida Guerra de Jesus for technical assistance. Patrícia de Oliveira receiveds cholarship from CAPES (Coordenação de Aper- feiçoamento do Pessoal de Nível Superior).